Implementation of a continuous, enzyme-coupled fluorescence assay for high-throughput analysis of glutamate-producing enzymes

Enzymatic formation of glutamate is critical to numerous biological pathway s. However, current methods for assaying the activities of glutamate-formin g enzymes are not particularly suitable for high-throughput screening in dr ug discovery. We present a continuous-read, fluorometric assay for high-thr oughput analysis of glutaminases. This assay is adapted to a microplate for mat and employs glutamate oxidase and horseradish peroxidase to couple glut amate formation to production of the fluorescent reporter molecule, resoruf in, for enhancement of sensitivity (M. Zhou, Z. Diwu, N, Panchuk-Voloshina, and R. P. Haughland, 1997, Anal. Biochem. 253, 162-168). Described herein is the selection of suitable levels of coupling enzymes for optimal kinetic response and lag time of the reporter system, based on the kinetic charact eristics of the individual coupling enzymes. Finally, implementation of the assay in a format for high-throughput kinetic analysis of glutaminases is demonstrated for Escherichia coli carbamoyl phosphate synthase. Derived kin etic constants are comparable to literature values determined using a varie ty of assay techniques. (C) 2000 Academic Press.